Stably overexpressed human Frizzled-2 signals through the beta-catenin pathway and does not activate Ca2+-mobilization in Human Embryonic Kidney 293 cells.
Signal transduction via the Frizzled family of seven-transmembrane receptors controls important developmental processes. Aberrant signaling caused by altered Frizzled receptor activity or by mutations in downstream signaling components has been implicated in several adult pathologies. A diverse array of intracellular signaling pathways has been suggested to transduce the signals exerted in cells when secreted ligands of the Wnt family bind to Frizzled receptors. Studies with a chimeric receptor composed of Frizzled-2 and the beta2-adrenergic receptor have suggested that the binding of Wnt-5a to Frizzled-2 results in the activation of G proteins of the Galpha(i) family, the mobilization of calcium from intracellular stores and the induction of gene transcription through nuclear factor of activated T cells. In this report, we demonstrate by using beta-lactamase reporter gene technology that full-length, wild-type human Frizzled-2 does not couple to calcium-mediated signaling in HEK293 cells following stimulation with purified recombinant mouse Wnt-5a. In contrast, when stimulated with recombinant mouse Wnt-3a, Frizzled-2 activates the canonical Wnt/Frizzled signaling pathway, involving the transcriptional modulator beta-catenin. Our report underlines the importance of using cell lines stably overexpressing wild-type Frizzled receptors and the use of purified ligands when studying receptor pharmacology. This approach has allowed us to measure the half-maximal concentration for activation of human Frizzled-2 (1.5+/-0.4 nM; avg.+/-SD) and human Frizzled-1 (1.3+/-0.5 nM) following stimulation by Wnt-3a. Our results suggest that there is receptor redundancy with regard to Wnt-3a reception. In addition, we introduce beta-lactamase reporter gene technology as an alternative to luciferase-based reporters to measure Frizzled receptor modulation for the discovery of Frizzled receptor-interacting drugs.
Verkaar
Folkert
F
Schering-Plough Research Institute, Molecular Pharmacology Unit, PO Box 20, 5340 BH Oss, The Netherlands.
van Rosmalen
Jos W G
JW
Smits
Jos F M
JF
Blankesteijn
W Matthijs
WM
Zaman
Guido J R
GJ
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D051157:Frizzled Receptors / Q000378:metabolism*; D017930:Genes, Reporter; D006801:Humans; D008163:Luminescent Measurements; D051379:Mice; D010405:Penicillinase / Q000378:metabolism; D051381:Rats; D018343:Receptors, Adrenergic, beta-2 / Q000378:metabolism; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D051153:Wnt Proteins / Q000378:metabolism; D051176:beta Catenin / Q000378:metabolism*
C489742:FZD2 protein, human; D051157:Frizzled Receptors; D018343:Receptors, Adrenergic, beta-2; D043562:Receptors, G-Protein-Coupled; D051153:Wnt Proteins; D051176:beta Catenin; D010405:Penicillinase; D002118:Calcium